This invention concerns enzyme-mediated chemical reactions conducted in vitro, and techniques for preventing their inhibition in the presence of detergents. Detergents are common tools in medical and biological research laboratories, primarily due to their ability to solubilize various proteins, cell wall and membrane components, and other cellular organelles, substructures, and components which are either insoluble or incompletely soluble in aqueous solution alone. Thus, detergents are often components of extraction or lysis buffers, both as lytic agents and as effective inhibitors of undesired enzyme activities such as those contributed by the proteases and nucleases normally present in a crude cell lysate. Additionally, such detergents are often used for the same purposes in the purification of nucleic acids.
Most enzymes used as tools in applied molecular and biological chemistry are quite sensitive to detergents, especially strong ionic detergents such as sodium dodecyl sulphate (SDS) or lithium lauryl sulphate (LLS). Such ionic detergents can bind strongly to proteins, often resulting in irreversible denaturation of the protein. (See American Society for Microbiology, Manual of Methods for General Bacteriology 57-58 (1981) ). However, for precisely this reason ionic detergents such as LLS are often an extremely valuable and inexpensive short- to medium-term preservative of nucleic acids in solution. Thus, such agents are useful to assist in accomplishing the first step of a nucleic acid hybridization assay using microorganisms; extraction of the nucleic acids from microbial cells or particles. Ionic detergents help to solubilize the cell wall and cell membrane, and to simultaneously prevent degradation of the nucleic acids by nucleases. (See id.) Moreover, strong ionic detergents such as SDS or LLS are often added to the lysis, permeabilization, or transport media in which clinical specimens are conveyed to the laboratory for analysis.
Often nucleic acids obtained from a biological sample are subsequently subjected to enzymatic manipulation, such as digestion with a restriction endonuclease or an exonuclease specific to DNA or RNA. Additionally, nucleic acids obtained from such samples are often not present in amounts large enough for them to be directly detected and/or quantified by nucleic acid hybridization techniques. Thus, the nucleic acid sequences of interest in such samples must normally be enzymatically amplified to be detected.
In biological or clinical samples to be subjected to one or more rounds of nucleic acid amplification or another enzyme-mediated reaction, the detergent must be separated from the nucleic acids in solution before an enzyme can be added to the reaction mixture. Dialysis or ultrafiltration, which usually works well to remove small molecules from a solution will not effectively remove most detergents, probably due both to the size of the micelles formed by the aggregation of the detergent molecules, as well as ionic or hydrophobic binding of the detergent molecules to larger solutes. Moreover, neither dialysis nor ultrafiltration is conveniently adaptable for use in a commercial diagnostic kit.
It would be convenient and cost-effective to perform an enzyme-mediated reaction such as nucleic acid amplification or a restriction digest in the same tube or collection vessel as is used to transport the biological sample to the laboratory for analysis, i.e. in the presence of SDS or LLS. Alternatively, it would be desirable to conduct such a reaction using such a sample as the immediate starting material, rather than having to subject the sample to an additional detergent-removing step. Although SDS can be precipitated with solvents such as acetone, acetone can denature or precipitate some enzymes. Moreover, the desired reaction may be inhibited by traces of acetone or other precipitating agents.
Currently, nucleic acids in a crude sample are generally purified prior to conducting an amplification by means of a phenol/chloroform extraction and subsequent ethanol precipitation. The method of the present invention takes advantage of both the similarities and the differences between ionic and non-ionic detergents to eliminate the necessity for such a step, thereby allowing enzyme-mediated reactions to be performed using nucleic acids in a biological sample, even when the sample contains an amount of ionic detergent which would normally inhibit the reaction.
The present invention is preferably a method for performing a nucleic acid amplification reaction, such as the polymerase chain reaction (PCR) or a transcription-based amplification system, in the presence of anionic detergents such as sodium dodecyl sulphate (SDS) or lithium lauryl sulphate (LLS). However, this present invention should be capable of preventing the inhibition of other enzymatic reactions, such as restriction digests, endo- and exonuclease digests, and kinase and transferase reactions by ionic detergents as well. Nor does the Applicant contemplate that the application of the present invention is limited to enzymatic reactions involving nucleic acids. Thus, while the embodiments of the present invention contained herein illustrate the use of the present invention in amplification reactions, such embodiments are meant to be exemplary only, the scope of the present invention being defined solely by the claims with which this specification concludes.
While not wishing to be bound by theory, Applicants believe that the formation of colloidal aggregates comprising heterogeneous micelles of non-ionic and ionic detergent molecules effectively remove the ionic detergent molecules from solution, thus making them unavailable to bind with or denature the subsequently added enzyme.
The ability of detergents to enhance or restore the activity of some enzymes has been reported. Saito, M., et al., Action of Arthrobacter ureafaciens Sialidase on Sialoglycolipid Substrates, 254 J. Biol. Chem. 7845-54 (1979). The use of heterogeneous micelles of ionic and non-ionic detergents as a method for the reactivation of detergent-inhibited proteins has also been reported. See Ey, P. L. & Ferber, E., Calf Thymus Alkaline Phosphatase II. Interaction with Detergents, 480 Biochim. Biophys. Acta 163-77 (1977) ; Berge, R. K., et al., Variations in the Activity of Microsomal Palmitoyl-CoA Hydrolase in Mixed Micelle Solutions of Palmitoyl-CoA and Non-Ionic Detergents of the Triton X Series, 666 Biochim. Biophys. Acta 25-35 (1981), Tandon S., & Horowitz, P. M., Detergent-assisted Refolding of Gaunidinium Chloride-denatured Rhodanese, 262 J. Biol. Chem. 4486-91 (1987). Additionally, the formation of heterogeneous micelles of ionic and non-ionic detergents has been reported as a method for removing inhibiting concentrations of non-ionic detergents from a solubilized enzyme preparation. Stralfors, P. et al., Removal of Nonionic Detergent from Proteins Fractionated by Electrofocusing, 533 Biochim. Biophys. Acta 90-97 (1978).
All of the methods mentioned in the publications listed above involve the activation or reactivation of enzymes in a detergent solution; none of these methods teach or suggest the formation of heterogeneous micelles in a solution containing an enzyme substrate before the addition of active enzyme. Additionally, in all these cases detergents were used to solubilize, purify, or activate the enzyme; in no case was the detergent initially added to solubilize and stabilize the enzyme substrate rather than the enzyme itself.